Three methods, i.e., structural analysis using a two-dimensional crystal based on electron crystallography, single particle analysis, and electron tomography, are known as methods of performing structural analysis of biological samples using an electron microscope.
In a structural analysis using a two-dimensional crystal based on electron crystallography, three-dimensional structures are analyzed by obtaining electron diffraction patterns and electron microscope images (bright-field images) from a sample tilted at various tilt angles (e.g., 0°, 20°, 45°, 60°, and so on), calculating amplitude data from the intensities of the electron diffraction patterns at diffraction points, and calculating phase data from the electron microscope images. Where a good two-dimensional crystal is obtained, the use of electron crystallography allows for analysis up to a high resolution of 1.9 Å. At such high resolution, the structure of membrane proteins as well as detailed structures including lipid molecules and water molecules can be analyzed.
The method of single particle analysis is a technique of reconstructing the three-dimensional structure of a molecule by image processing from electron microscope images of the particles of isolated biological macromolecules, such as protein molecules (see, for example, patent literature 1).
In methods of single particle analysis, the structures of membrane proteins such as TRP (Transient Receptor Potential) channels have been structurally analyzed in recent years at a resolution of 3.4 Å. In single particle analysis, three-dimensional structures of membrane proteins can be analyzed at such a high resolution that an atomic model can be generated without crystallization.
The development of methods of single particle analysis of recent years has been brought about by the revolutionary development in an apparatus for recording images, as well as by development of stable cryoelectron microscopes. One technique had been employed is to record electron microscope images on film. Another technique consists of converting an electron beam into light by a fluorescent agent and recording the light with a camera using CCDs (charge coupled devices) or the like. On the other hand, in recent years, there has been developed an apparatus wherein an electron beam is directly recorded in a CMOS (Complementary MOS) camera or the like, and the recording method has been improved. This has greatly improved both the DQE (Detectable Quantum Efficiency) and the MTF (Modulation Transfer Function). Owing to such instrumental development, it has become possible to record high-resolution images at extremely high efficiency. Such technical evolution has allowed for high-resolution structural analysis of even membrane proteins without producing crystals by the use of a method of single particle analysis.
However, in structural analysis of TRP using single particle analysis, there is the problem that, if detergents are used, micelles create serious background noise. Therefore, in structural analysis of TRP, micelles have been removed by replacing the surfactant by amphipols. In the case of TRP, the surfactant can be successfully replaced by amphipols but this replacement is not always generally feasible.
Electron tomography is a technique of reconstructing a three-dimensional structure by tilting a sample in small angular increments (i.e., tilting it almost continuously), taking a number of electron microscope images, and image processing them (see, for example, patent literature 2). If electron tomography is used, three-dimensional morphologies of complex structures can be analyzed. However, it is difficult to improve the SN ratio by averaging many molecules such as methods of structural analyses of single particle analysis and electron crystallography. Under the present situation, therefore, it is difficult to make analyses at resolutions higher than 30 Å.
As described so far, three kinds of methods are used for high-resolution structural analysis using an electron microscope. Especially, methods of single particle analysis are attracting much attention. The reason why methods of single particle analysis are attracting attention is that a three-dimensional structure can be analyzed even if an effort to achieve crystallization, which is not certain to succeed, is not made and that analysis at resolutions higher than 3.5 Å is possible, it being noted that at this resolution of 3.5 Å, an atomic model can be created. Consequently, methods of single particle analysis have received much expectation and attention in research areas including many applications typified by structural researches for drug discovery, as well as in development of fundamental biological researches.